Monoradioiodinated imidazole derivatives

ABSTRACT

Monoradioiodinated derivatives of compounds employed in a radioassay prepared from precursors which are either active esters, amino acids, or amines, including a phenolic of imidazole substituent group in which one of the possible two sites on the group for radioiodination is substituted to permit production of a monoradioiodinated derivative. A preferred precursor is an active ester of 3-fluoro-5-radioiodotyrosine which can be coupled to a compound including an amino group to produce a monoradioiodinated derivative of the compound.

This is a division of application Ser. No. 885,447, filed Mar. 10, 1978,now U.S. Pat. No. 4,202,874, which is a division of application Ser. No.727,407 filed Sept. 29, 1976, now U.S. Pat. 4,120,867.

This invention relates to mono-radioiodinated precursors, intermediatesemployed for the preparation thereof and compounds having the precursorscoupled thereto.

In a radioimmunoassay, the compound including a radioactive isotope ofiodine is generally a compound which has the radioiodinated isotopesubstituted on a phenolic moiety. In the preparation of suchradioiodinated compounds by conventional techniques, in general, thereaction mixture includes monoiodo, diiode and unreacted derivatives. Inmany cases, it is advantageous to employ monoiodo derivatives and,accordingly, there is a need for a means for conveniently producing suchradioiodinated derivatives.

An object of the present invention is to provide monoradioiodinatedcompounds.

Another object of the present invention is to provide precursorssuitable for coupling to a compound to provide a radioiodinatedderivative of such compound.

A further object of the present invention is to provide radioiodinatedderivatives of compounds suitable for use in radioassays.

These and other objects of the present invention should be more readilyapparent from reading the following detailed description thereof.

In accordance with one aspect of the present invention, there areprovided precursors which are either unlabeled or radiolabeled with aradioactive isotope of iodine, with the unlabeled precursors having astructure such that radioiodination thereof produces only amonoradioiodinated derivative, and with the labeled precursors beingmonoradioiodinated. The precursors are further characterized bystructure such that they can be coupled to a compound including an aminoor carboxy group. The precursors of the present invention arerepresented by the following structural formula: ##STR1## wherein

R is a straight chain or branched chain divalent aliphatic hydrocarbonradical having from 1 to 6 carbon atoms;

X is hydrogen or an active ester moiety;

Z is either acyl or benzyloxy-carbonyl;

Y is either: ##STR2## wherein

one of R₁ and R₂ is either hydrogen or a radioactive isotope of iodineand the other of R₁ and R₂ is either lower (1-6 carbon atoms) alkyl,lower (1-6 carbon atoms) alkoxy, fluoro-, chloro-, bromo, or nitro-;

A is either hydrogen, lower (1-6 carbon atoms) alkyl, an alkali metal(preferably sodium) or an alkaline earth metal and n is 0 or 1;

the radioactive isotope is preferably ¹²⁵ I, although other radioactiveisotopes are also useful; e.g., ¹²⁷ I;

the compounds wherein one of R₁ and R₂ is a radioactive isotope ofiodine are novel precursors.

The preferred precursors are the monoradioiodinated derivatives and inparticular the active esters in that such precursors can be coupled to acompound, including an amino or carboxy group, to provide thecorresponding monoradioiodinated derivative of such compound.Alternatively, and less preferred, the unlabeled precursor can becoupled to a compound, including an amino or carboxy group, followed byradioiodination of the resulting derivative to produce the correspondingmonoradioiodinated derivative. The compounds having a precursor coupledthereto are represented by the following structural formulae: ##STR3##wherein B is lower alkyl and Y and R are as hereinabove described.

The compounds which are coupled with the precursors of the presentinvention, are preferably compounds for which an appropriate receptorcan be found or the receptor, itself. The compounds can be:

(1) antigens, which when introduced into the blood stream of avertebrate, result in the formation of antibodies;

(2) haptens, which when bound to an antigenic carrier and introducedinto the blood stream of a vertebrate, produce antibodies specific forthe hapten; or

(3) compounds which have naturally occurring receptors and can beisolated in a form specific for the compound, it is to be understoodthat the compounds can have naturally occurring receptors and alsofunction as a hapten when bonded to a protein.

Alternatively, as hereinabove noted, the antibody elicited in responseto the antigen or hapten bound to a protein, or the naturally occurringreceptor, can be coupled with the precursor material.

As representative compounds which can be coupled to the precursor inaccordance with the present invention, there may be mentioned:

[1] drugs, including alkaloids; e.g., opiates, such as morphine, heroinand the like; methadone and its analogs; indole alkaloids;catecholamines; barbiturates; glutethimide; cocaine and its metabolitesand analogs; diphenyl hydantein; marijuana; tranquilizers, e.g.meprobamate; phenothiazines, etc.:

[2] amino acids, polypeptides, nucleotides, nucleosides and proteins,such as ACTH, oxytocin, luteinizing, hormone, insulin, Bence-Jonesprotein, chorionic gonadotropin, pituitary gonadotropin, growth hormone,renin, thyroxine binding globutin, bradykinin, anglotensin, folliclestimulating hormone; thyroid stimulating hormone: cyclic AMP; etc.

[3] steroids, including: oestrogens, gestrogens, androgens,adrenocortical hormones, bile acids, cardiotonic glycosides, aglycones,as well as Saponins. As specific examples, these may be mentioned:testosterone, androsterone, equilenin, estrone, estriol, progesterone,pregnenolone, 17-hydroxy-deoxy-corticosterone (compound S),deoxycorticosterone, cortisone, corticosterone, cortisol, aldesterone,digoxigenin, disitoxigenin, etc.

[4] vitamins, such as Vitamin A, the B vitamin group, vitamin C, the Dvitamins, and vitamins E and N; folic acid and miscellaneous biologicalsubstances, such as, antibodies, e.g., penicillin, tetracycline;antigens for Viral Heptatis A and B, Rubella, Herpes Simplex, Alphafetoprotein, antibodies to N.gonorrhea, Dane Cores, etc.

The above substances are only representative, and it is understood thatif the compounds do not include an amino or carboxy group for effectingcoupling to the precursor the compound is employed as an appropriateanalog which includes an amino or carboxy group for effecting couplingto the precursor.

The unlabeled precursors of the present invention are either known inthe art or can be prepared by procedures known in the art. Thus, forexample, the substituted hydroxyphenyl substituted acids, in particular3-fluoro-4-hydroxyphenylpropionic acid can be prepared by converting3-fluoro-4-methoxybenzaldehyde to 3-fluoro-4-methoxy-cinnamic acid,which can be reduced to 3-fluoro-4-methoxyphenylpropionic acid, followedby hydrolysis.

The active ester precursors are prepared by procedures known in the art.The active ester may be any one of a wide variety of active esters whichare suitable for coupling the precursor to an amino group. The activeesters are well-known in the art; for example, such esters are describedin "Recent Trends in the Synthesis of Linear Peptides" by A. Kapoor J.Pharm. Science 1970 59 (1) pages 1-27, and the selection of anappropriate active ester is deemed to be within the scope of thoseskilled in the art from the teachings herein. The preferred activeesters are prepared from chloro- or nitro substituted phenols orN-hydroxysuccinimide; however, the invention is not limited to suchpreferred groups.

The precursors are coupled to the compounds by procedures known in theart. For example, the active ester precursors are coupled by reaction ofthe active ester precursor with the compound in a suitable buffer (pH6-9) at temperatures of from -10° to 15° C. The precursors which do notcontain active ester groups can be coupled by the use of a suitablecoupling agent; e.g., dicyclohexylcarbodiimide, or by the mixedanhydride technique. A glycoside, such as digoxin, may be coupled to theprecursor by opening the sugar portion of the glycoside by the use ofperiodate, followed by coupling of the resulting dialdehyde to an aminoprecursor, with such a technique generally being described by Haber,Biochemistry Vol. 9, No. 2, Jan. 20, 1970.

The technique for coupling the precursors to produce the coupledcompounds of the present invention are well known in the art and nofurther details in this respect are required for a completeunderstanding of the present invention.

The compounds including the coupled monoradioiodinated substituent aresuitable for use in a radioassay by procedures known in the art. Thus,the coupled radioiodinated compounds of the present invention may beemployed for a radioassay in a manner identical to the radioiodinatedcompounds employed in prior art radioassays.

The assay employing the monoradioiodinated coupled compound is effectedby general procedures known in the art, which involve:

[1] combining receptor, sample, and monoradioiodinated labeledsubstance;

[2] determining the radioactive properties of the bound or free portion;and

[3] comparison of such properties with a standard.

Prior to determining the radioactive properties of the bound or freeportion, it is preferred to effect separation of such bound and freeportions by procedures known in the art. Thus, for example, suchseparation may be effected by the use of solid adsorbents, gelfiltration, ion exchange resins, etc. Alternatively, the receptor may bebound to a suitable substrate to facilitate separation.

The receptors employed in the assay, as known in the art, are, in themost part, macromolecules which recognize specific structures, with suchreceptors generally being proteins and nucleic acids which are found incell membranes, blood and other biological fluids.

The most generally used group of receptors are antibodies, which areconveniently used in the assay of haptens and antigens. The antibodiesare produced by introducing an immunogenic substance into thebloodstream of a living animal and there are many materials which act asantigens to produce an immunogenic response. Haptens can also beemployed to prepare antibodies by procedures well known in the art. Ingeneral, such a procedure involves bonding the hapten to a protein,followed by introduction thereof into the bloodstream to produceantibodies for the hapten; i.e., the compound bonded to the protein. Theantibody thus generated, as known in the art, can be employed as areceptor in an assay for the hapten.

In addition to antibodies, there are many naturally occurring materialswhich are specific to compounds of biological interest and can be usedin the assay of the present invention. Thus, for example, as known inthe art, there are naturally occurring receptors which are suitable forthe assay of materials, such as, folates, thyroxine, corticosterone,cortisone, estrogen, insulin, angiotensin.

The use of receptors in an assay for various compounds is well known inthe art and no further details in this respect are deemed necessary foran understanding of the present invention.

The invention will be further described with respect to the followingexamples; however, the scope of the invention is not to be limitedthereby:

EXAMPLE 1 I-Preparation of 3-fluoro-4-methoxy-cinnamic acid

A mixture of 3-fluoro-anisaldehyde (1.5 g) and malonic acid (2.08 g) in5 ml pyridine was warmed until clear solution was obtained. To thismixture piperidine (0.4 ml) was added and then the content of the flaskwas heated at 80° C. for one (1) hour. It was further refluxed for 3hours. At the end of this period the mixture was poured into 50 ml ofcold water. It was then acidified by adding 5 ml of concentratedhydrochloric acid. Upon addition of acid, the solid separated, which wasfiltered and washed with cold water. The material was dried. Yield=1.7g. The crude material was crystallized from a mixture of tetrahydrofuranand water. m.p. 227°-230° C.

II-Preparation of 3-(3-fluoro-4-methoxyphenyl)propionic acid

2.0 g of 3-fluoro-4-methoxy cinnamic acid was dissolved in 100 ml ethylalcohol. To this was added 100 mg of platinum oxide as a catalyst. Themixture was hydrogenated in a Parr apparatus for 90 minutes. After thehydrogenation was over, the mixture was filtered and concentrated. Thesolid was crystallized by adding water. Yield=1.63 g. m.p. 107°-112° C.

III-Preparation of 3-(3-fluoro-4-methoxyphenyl)propionic acid

To 1.0 gm of 3-fluoro-4-methoxyphenyl propionic acid (II) in a 50 mlround bottom flask, 15.0 ml of III (47-51%) was added and the mixturewas heated at 140° C. for 1 hour. At the end of 1 hour, the mixture wasconcentrated and 25 to 30 ml water was added. It was again concentratedusing a rotary evaporator. Finally 50 ml water was added and the pH wasadjusted to 3 by adding sodium bicarbonate. The aqueous mixture was thenextracted with benzene and the benzene layer was washed twice withwater. Finally, the benzene layer was dried over anhydrous sodiumsulfate. The benzene was evaporated off. The product was in the oilyform and was used in the next step for the preparation of active ester.

IV-Preparation of N-succinimidyl-3-(3-fluoro-4-hydroxyphenyl) propionate

3-(3-fluoro-4-hydroxyphenyl)propionic acid (1.03 g) andN-hydroxysuccinimide (1.15 g) in tetrahydrofuran (7.0 ml) was treated at-18° C. with dicyclohexylcarbodiimide (2.47 g). The mixture was stirredat -18° C. for 2 hours, kept at room temperature for 10 hours andtreated with acetic acid (0.12 ml) to destroy excess of carbodiimide.After 1 hour, the mixture was diluted with ethyl acetate (10 ml). Thedicyclohexyl urea was filtered. The ethyl acetate layer was evaporatedto dryness. The residual oil was then crystallized from ethylacetate/petroleum ether. m.p. 100°-102° C.

V-Radioiodination of active ester

The reaction is carried out at room temperature (about 20° C.)N-succinimidyl-3-(3-fluoro-4-hydroxylphenyl)propionic acid (0.2 to 0.025mg) was treated with 2 to 5 mCi of NaI¹²⁵ and 50 μg of chloramine-T in10 μl of 0.25 M phosphate buffer pH 7.5. The reaction was immediatelyterminated by the addition of 120 μg of sodium metabisulfite in 10 μl of0.05 M phosphate buffer, pH 7.5, after which 200 mg of carrier K1 in 100μl of the above buffer was added. The reaction mixture was extractedinto two portions of 0.25 ml of dry benzene containing 10 μl ofdimethyl-formamide. This solution was then transferred into a V-shapedglass vial containing a magnetic flea for further use.

VI-Coupling of I¹²⁵ -active ester to thyroid stimulating hormone (TSH)

2.5 mCi of I¹²⁵ active ester (step 5) was transferred into a V-shapedvial containing a magnetic flea and the vial was sealed using ateflon-septum cap. The benzene was then evaporated by passing a slowstream of N₂ gas while trapping any volatile radio activity in anattached charcoal trap. The cap from the vial was removed and added 10μl (5 μg) of hTSH (human TSH) solution and 2 μl of 0.5 M sodium boratebuffer pH 8.5. The mixture was stirred at 4° C. for 20 minutes. 10 μl of0.1 M sodium borate buffer pH 8.5 was added to the reaction vial andstirred at 4° C. for 5 minutes. 0.5 ml of 0.2 M glycine solution wasadded to the reaction mixture and stirred at 4° C. for an additional 5minutes. At this point, the reaction is over. This mixture wastransferred to a Sephadex-G-75 column (size 90×1.7 cm) and the columnwas eluted at 4° C. with 0.05 M sodium phosphate buffer (pH 7.5)containing 0.25% gelatin. The appropriate peak for hTSH was separatedand stored separately for further use in radioimmunoassay.

EXAMPLE 2 Testosterone 3-(0-carboxymethyl)oxime-3-fluoro-tyrosinemethylester I¹²⁵

(a) Preparation of 3-fluoro-tyrosine-I¹²⁵

Three milligrams of 3-fluoro-tyrosine HCl were dissolved in 5 mlphosphate buffer (0.5 M, pH 7.5). Ten microliters of this solution in asuitable vial were treated with 5 mCi of NaI¹²⁵ followed by 10 μl ofchloramine-T solution of 50 mg/10 ml concentration. After 60 seconds, 10μl of sodium metabisulfite solution of concentration 300 mg/10 ml wasadded to terminate the reaction.

(b) Steroid activation

To 2.5 mg of testosterone-3-(0-carboxymethyl)oxime in a tube, 50 μl ofdioxane, 10 μl of prediluted tributylamine (1 ml t-butylamine/9 mldioxane) and 10 μl of prediluted isobutyl chloroformate (1 ml ofisobutyl chloroformate/3 ml dioxane) were added. The reaction mixturewas stirred at 10° C. for 20 minutes.

(c) The solution in (b) was diluted to 3.43 ml using dioxane and 50 μlof this diluted solution was added to the solution in (a) followed by 10μl of 0.1 N NaOH solution. The entire reaction mixture was maintained at0° C. for 2 hours. The reaction mixture was then acidified with 0.1 NHCl (0.9 ml) and extracted with 1 ml toluene. The organic layer wasdiscarded. To the aqueous layer, 0.9 ml of 0.1 N NaOH and 1 ml of 0.5 Mphosphate buffer (pH 7.0) were added. The aqueous layer was extractedwith ethyl acetate. The ethyl acetate layer contained the final product.

EXAMPLE 3 Testosterone-17-β-0-succinyl-3-fluoro-tyrosine methyl ester

(a) A mixture of 7.5 mg of testosterone-17-β-hemisuccinate, 6 mg3-fluoro-tyrosine methyl ester. HCl and 6 mg of EDC(1-ethyl-3-(dimethylaminopropyl carbodiimide. HCl) in a vial containing0.3 ml tetrahydrofuran and 0.05 ml water, was stirred at the roomtemperature for 90 minutes. Then it was purified by preparativechromatography using silica gel plates. Solvent system, CHCl₃ :MeOH:H₂O=180:20:0:2. The product, R_(f) value of 0.52, was isolated byextracting the silica gel zone with ethanol.

By using a similar procedure, several other derivatives were made, e.g.,testosterone-17-β-0-succinyl-3-fluoro-tyrosine ethyl ester andtestosterone-17-β-3-fluoro-tyrosine-t-butyl ester.

(b) Preparation of testosterone-17-β-0-succinyl-3-tyrosine methylester-I¹²⁵

The product (a) described above was iodinated as follows:

The alcoholic solution of product (a) 10, mole in 10 μl was taken into asmall vial containing 50 μl phosphate buffer (0.5 M pH 7.5). To this 25μl of chloramine-T solution was added (10 mg/3 ml) followed by 5 μl ofNaI¹²⁵. After 30 seconds the reaction was terminated by adding 25 μl ofsodium metabisulfite solution (30 mg/3 ml). The product was purified bythin layer chromatography using silica gel coated plates and running itin CHCl₃ :MeOH:H₂ O=180:20:0:2 as a solvent system.

EXAMPLE 4 Aldosterone-3(0-carboxymethyl)oxime-3-fluoro-tyrosine methylester-I¹²⁵

(a) Steroid activation

To 1.5 mg of aldosterone-3(0-carboxymethyl)oxime in a tube, 50 μl ofdioxane was added as a solvent. To the above reaction mixture 10 μl ofprediluted tributylamine (1 ml tributylamine/15 ml dioxane) and 7 μl ofprediluted isobutyl chloroformate (1 ml of isobutyl chloroformate in 6ml dioxane) was added and the reaction mixture was stirred at 10° C. for20 minutes.

(b) Coupling

The solution (a) from above reaction was diluted by adding 1 ml ofdioxane. 50 μl of this diluted solution was added to solution (a) ofExample 2 followed by the addition of 10 μl solution of 0.1 N NaOH. Theentire reaction mixture was maintained at 0° C. for 2 hours. Thereaction mixture was then acidified with 0.1 N HCl (0.9 ml) andextracted with 1 ml toluene. The organic layer was discarded. To theaqueous layer was added 0.9 ml of 0.1 N NaOH and 1 ml of 0.5 molarphosphate buffer (pH 7.0). The aqueous layer was extracted with ethylacetate. The ethyl acetate layer contained the final product.

EXAMPLE 5 Digoxin-3-fluoro-tyrosine methyl ester-I¹²⁵

600 mg of digoxin was suspended in 30 ml of absolute ethanol. To thissuspension was added slowly 30 ml of 2% sodium periodate in water. Thereaction mixture was stirred for 1 hour. After 1 hour, it was evaporatedto dryness. The residue was dissolved in 5.0 ml water and then extractedwith ethyl acetate. The ethyl acetate layer was dried over sodiumsulphate and evaporated to dryness to yield the solid dialdehyde. 250 mgof the dialdehyde was dissolved in 10 ml methanol. To this,3-fluoro-tyrosine methyl ester (65 mg) and 50 mg of sodiumcyanoborohydride was added. The pH of the reaction mixture was about7.3. The reaction mixture was stirred for 90 minutes at roomtemperature. The pH was lowered from 8.2 to 7.2 by adding 0.05 N HClsolution. The reaction mixture was concentrated to dryness, 10 ml ofwater was added and the aqueous layer was then extracted with ethylacetate. The ethyl acetate layer was dried and concentrated. The productwas crystallized from ethyl acetate/ether.

Radioiodination

To 2.0 mg of the above product in 20 ml of methanol in a vial, 5 mCiNaI¹²⁵ was added followed by 100 μg of chloramine-T. The reaction wasterminated by adding 300 μg of sodium metabisulfite solution. Theproduct was purified by thin layer chromatography using silica gelplates. Solvent system, ethyl acetate:methanol=96:4.

EXAMPLE 6 (a) Preparation of cholylglycyl-3-fluoro-tyrosine methyl ester

A mixture of 46 mg of cholylglycine, 25 mg of 3-fluoro-tyrosine methylester and 65 mg of EDC (1-ethyl-3-(dimethylamino propyl) carbodiimideHCl was stirred magnetically in 300 μl tetrahydrofuran and 75 μl ofwater. The stirring was stopped after 2 minutes. The mixture wasextracted with 3 ml ethyl acetate. The ethyl acetate layer was washedwith water, dried over sodium sulfate and then concentrated to dryness;yield=40 mg, R_(f) 0.44, solvent system CHCl₃ :MeOH 8:2.

(b) Preparation of cholylglycyl-3-fluoro-tyrosine (saponification ofproduct 6(a).

A mixture of cholylglycyl-3-fluoro-tyrosine methyl ester (Example 6(a)),200 μl dioxane and 200 μl of 2.5 N NaOH was stirred for 30 minutes atroom temperature. The pH was adjusted to 2 to 3 with 6 N HCl. Theaqueous layer was extracted with ethyl acetate. The ethyl acetate layerwas washed with water, dried over sodium sulfate and evaporated todryness.

(c) Preparation of I¹²⁵ Cholylglycyl-3-fluoro-tyrosine

Seven mg of cholylglycyl-3-fluoro-tyrosine was dissolved in 3.5 ml ethylalcohol and a 5 μl aliquot was mixed in a small vial with 50 μl of 0.5 Mphosphate buffer (pH 7.4) and 1.8 MCi of NaI¹²⁵. Ten (10) μl ofchloramine-T solution (15 mg/5 ml water) was added and, after 60seconds, the reaction was terminated by adding 10 μl of sodiummetabisulfite (25 mg/5 ml water). The reaction mixture was applied to aDowex 1×8 resin column. After washing with water, the product was elutedwith methanol containing 1% acetic acid.

EXAMPLE 7 Labeling of antibody

(a) Preparation ofN-succinimidyl-3(3-fluoro-4-hydroxyphenyl)propionate-I¹²⁵

N-succinimidyl-3(3-fluoro-4-hydroxyphenyl)propionate (1.32 mg) wasdissolved in 10 ml ethyl acetate. Twenty (20) μl of this solution wastransferred to a small vial and N₂ gas was slowly bubbled until all theethyl acetate had evaporated. To this residue, 20 μl of 0.5 M phosphatebuffer (pH 7.4) was added followed by 10 mCi of NaI¹²⁵ and 5 μl ofchloramine-T solution (25 mg/5 ml water). After 5 seconds, the reactionwas terminated by adding 10 μl of KI solution (100 mg/5 ml) and 5 μl ofdimethyl formamide. This mixture was extracted twice with 0.6 ml drybenzene. The benzene layer was separated, dried with anhydrous sodiumsulfate, and charged to a small column containing CaCO₃. The column waseluted with benzene. The first 5.0 ml collected contained the pureproduct.

(b) Coupling of active ester to antibody

0.25 MCi in 63 μl of benzene of the active ester I¹²⁵ in Example 7(a)was evaporated with N₂ gas. The vial was cooled in icewater and 1 ml ofrabbit IgG solution (1 mg of IgG/1 ml 0.1 M borate buffer pH 8.5) wasadded to the vial. The solution was stirred at 4° C. for 25 minutes andthen treated with 0.5 ml of 0.2 M glycine solution. The reaction mixturewas stirred for 10 minutes at 4° C. and the content of the tube wascharged to a Sephadex G-25 column at 4° C. The column was eluted with0.1 M phosphate buffer (pH 7.4) containing 0.1% BSA and 0.02% sodiumazide. The rate of column elution was adjusted to 10 ml per hour and 1ml volume fractions were collected. The product was eluted at fractionnumbers 68 to 74.

The present invention is particularly advantageous in that it ispossible to easily produce monoradioiodinated derivatives of compoundswhich can be used as the tagged or labeled compound in a radioassay.

A monoradioiodinated derivative is advantageous, as compared todiradioiodinated derivatives, in that the monoradioiodinated derivativeshave improved stability and longer half-lives. In addition, themonoradioiodinated derivatives are altered less and for a given specificactivity insures good physiological parameters. In addition, theprecursors can be prepared with an excess of the radioactive isotope tominimize production of unlabeled precursor, without production of diiododerivative.

In addition, the radioiodination can be effected with two differentradioisotopes; e.g., ¹²⁵ I and ¹²⁷ I, and by adjusting the ratiothereof, it is possible to obtain a desired specific activity.

These and other advantages should be more apparent from the hereindescription of the present invention.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

What is claimed is:
 1. A compound selected from the group consisting of:##STR4## wherein R is selected from the group consisting of straight andbranched chain aliphatic hydrocarbons having from 1 to 6 carbon atoms;Xis selected from the group consisting of hydrogen and active esters forcoupling the compound to an amino group; Z is benzyloxycarbonyl; Y is##STR5## wherein one of R₁ and R₂ is a radioisotope of iodine and theother of R₁ and R₂ is selected from the group consisting of loweralkoxy, fluoro-, chloro-, bromo-, and nitro-; A is selected from thegroup consisting of hydrogen, lower alkyl, an alkali metal and analkaline earth metal.
 2. The compound of claim 1 wherein the compoundhas structural formula (a).
 3. The compound of claim 1 wherein thecompound has structural formula (b).
 4. The compound of claim 1 whereinthe compound has structural formula (c).
 5. The compound of claim 1wherein one of R₁ and R₂ is fluorine and the other of R₁ and R₂ is aradioactive isotope of iodine.
 6. The compound of claim 1 wherein theradioactive isotope is ¹²⁵ I.
 7. A compound selected from the groupconsisting of: ##STR6## wherein R is selected from the group consistingof straight and branched chain aliphatic hydrocarbons having from 1 to 6carbon atoms;X is an active ester moiety for coupling the compound to anamino group; Z is benzyloxycarbonyl; Y is ##STR7## wherein one of R₁ andR₂ is a radioisotope of iodine and the other of R₁ and R₂ is selectedfrom the group consisting of lower alkyl, lower alkoxy, fluoro-,chloro-, bromo-, and nitro-.
 8. The compound of claim 7 wherein one ofR₁ and R₂ is lower alkyl and the other of R₁ and R₂ is a radioisotope ofiodine.
 9. The compound of claim 7 wherein one of R₁ and R₂ is fluorineand the other of R₁ and R₂ is a radioactive isotope of iodine.
 10. Thecompound of claim 7 wherein the radioactive isotope is ¹²⁵ I.
 11. Thecompound of claim 7 wherein the active ester is prepared from a memberselected from the group consisting of chloro- and nitro- substitutedphenols and N-hydroxysuccinimide.